1. Field of the Invention:
The present invention is directed to a method of detecting the presence or concentration of an analyte in a sample solution. More particularly, the invention is directed to a method of improving the sensitivity and substantially eliminating memory effects in analytical detection methods by premixing the sample solution with a particulate reagent such that the analyte is bound to the particulate reagent prior to analysis.
2. Discussion of the Background
Analytical methods are employed to analyze a wide variety of sample types. Often, the initial chemical or physical form of a sample is incompatible with the analytical method or instrument and so the sample must be changed or treated before analysis. For example, solid samples are often dissolved to form an aqueous solution prior to analysis. A number of other sample treatments are also used to enhance the analysis process, including treatments to concentrate analytes, reduce sample matrix interferences or to improved sample introduction ease or efficiency. Ideally, these treatments result in faster, more sensitive and more accurate analysis.
The sensitivity of an analytical method is of critical importance. A sample may contain analyte concentrations that are of interest, but are still lower than the detection limits of the system used to analyze them. Inadequate instrument sensitivity can be caused by lack of an adequate amount of analyte, by low analyte concentrations or by the presence of interfering substances. In these cases, the apparent sensitivity of the instrument can be improved by better methods of sample introduction, preconcentration or removal of interfering substances.
For example, the determination of trace metals at low levels in drinking water is a common analytical problem. Current instrumentation, e.g., flame atomic absorption spectrometry, may not obtain the desired sensitivity. A rapid on-line continuous preconcentration method would allow the desired detection with improved sensitivity while maintaining high sample throughout.
Preconcentration of a sample may be accomplished by partial evaporation of the solvent. However, evaporation methods are not on-line methods and are generally extremely slow. Furthermore, evaporation is not selective. All materials that are not volatile are concentrated, whether these materials are of interest or not. Volatile materials are lost, whether they are of interest or not.
Numerous sample treatment and preconcentration methods are known, where the sample is passed through a packed bed of material in a column. The column may retain an interfering substance and let the analyte pass through the column to be analyzed, or alternatively, the column may retain and preconcentrate the analyte and let the sample matrix pass through the column to waste. See for example U.S. Pat. No. 4,357,143.
In a typical column application, transition metal ions are passed through a column containing a chelating ion-exchange resin. Analyte species are bound to the column while solvent and unwanted matrix components such as sodium ions pass through the column as waste. The column bound transition metal are then released from the column packing resin and washed from the column using an eluant. The eluting liquid usually has a different pH or polarity than the sample so that the analyte is released from the packing resin. Eventually, the analyte is transported to an instrument for analysis. Column preconcentration and matrix removal methods are subject to several limitations including reversible complexation effects, poor kinetic effects, increased analysis time and contamination.
To perform a preconcentration process using a column, the solid particles which chemically bind the analyte, e.g., by ion exchange, chelation, adsorption, etc., must be able to release the analyte species prior to analysis. Thus, packing resins which appear to be most suited to perform the preconcentration process because they bind the analyte of interest strongly and selectively, may not be useful because it is difficult or impossible to release the analyte from the column resin with a reasonable volume of eluant. Additionally, incomplete elution of analytes can cause memory effect problems.
Fast kinetics are desirable for column preconcentration operations. The best kinetics occur on the surface of the column packing material. Therefore, large surface area to volume ratios, i.e., small particles, are desirable for the column packing material. However, small suspension particles cannot be packed easily into a column without causing high fluid back pressures. Thus, the use of optimal particle sizes for binding analytes are not possible using preconcentration columns.
A preconcentration column must be loaded with sample and then the sample must be eluted from the column. This is a discontinuous process. Since the preconcentration column is usually used for many analyses, it must be cleaned and reconditioned between analyses. Accordingly, operation of a preconcentration/matrix removal column usually increases the analysis time.
The use of elution solvents introduces an additional step into the analysis process and may also introduce contamination into the sample eluted from the column. Furthermore, incomplete elution is one cause of memory effects. Residual material can elute causing a residual signal over a long period of time, i.e., a memory effect.
Another of the many problems which prevent accurate sensitive analytical analysis is the occurrence of memory effects. Memory effects are observed when residual analytes from a previous analysis are detected in a current analysis. More often, memory effects are observed from volatile elements that are adsorbed or retained through a variety of mechanisms within the components of an analytical device.
Memory effects are manifested in essentially two ways. First, the detected signal, from analyte trapped within the instrument and gradually released, may persist over a period of several minutes to several hours. This prolonged signal results in a high, changing background signal. As a result, the analysis of subsequent samples, especially those with low analyte concentrations, is difficult or impossible. A second manifestation of the memory effect is dependent upon the sample matrix. Some matrices may induce matrix-effected species to be retained within the analytical instrument. Other matrices may cause analytes that are retained within the instrument from previous samples to be released. In this way, depending on the sample matrix, the memory-effected element or analyte may be subject to either a false negative or a false positive signal.
Many chemical elements exhibit notorious memory effect behavior. For example, mercury is retained on the surface of sample introduction devices commonly used for atomic spectroscopy. Typically, a sample is aspirated into a nebulizer which converts the sample into an aerosol. The aerosol is then swept by a gas stream into an excitation source, such as an inductively coupled plasma (ICP) or a flame, where the analytical signal is generated. Mercury is adsorbed onto the walls of the nebulizer and transfer tubing where later, due to its high vapor pressure, it slowly bleeds into the aerosol stream, thereby producing the memory effect described above. Until the mercury has been completely rinsed from the system, samples with low concentrations of mercury cannot be accurately analyzed due to the high background signal. Complete rinsing of the system may take hours.
Many other elements such as arsenic, cadmium, gold, zinc and osmium exhibit memory effects as well. It would be desirable, therefore, to treat an analyte sample to eliminate memory effects for these and other analytes which exhibit a memory effect. Elimination of the memory effect is particularly important for analytical methods such as ICP-spectroscopy which are designed to determine the presence of multiple elements simultaneously. This advantage of ICP spectroscopy instrumentation is lost when mercury and other elements which exhibit a memory effect cannot be determined.
The uniform transport of analytes in an analytical device, is important to accurately to detect an analyte species. Analyte transport efficiency is degraded for analytes having high volatility (high vapor pressure). Such analytes exhibit a memory effect as well as non-uniform transport in the analytical device.
Many methods are known where a particulate reagent is added to an analyte solution. In some methods, the particulate reagent dissolves in the solution during the method. See, for example, DD 219,873 which describes a continuous flow method for determining HF and FeF.sub.2 /FeF.sub.3 where an aqueous suspension of MgO is added to the sample. The MgO is not insoluble, however, and dissolves in the solution during the method.
In other known methods, the particulate reagent is detected as part of the detection process. Many of these methods are related to immunoassay determinations of specific analytes or antigens. U.S. Pat. No. 4,650,770, for example, describes an immunoassay employing fluorescent particles and adsorbent particles. The adsorbent particles substantially inhibit fluorescence when bound to the fluorescent particles through specific non-covalent binding. The decrease in fluorescence of the insoluble particles is then measured.
DE 2,749,956 describes an immunoassay method using photometric detection of latex polymer reagents. Several measurements are made in this kinetic method which cannot be readily adapted to continuously flowing analyte streams. Japanese patent Nos. 59/171863, 62/002163, 62/093663 and 62/093664 are also directed to kinetic methods.
U.S. Pat. No. 4,665,020 describes a flow cytometric measurement of a binding competition immunoassay where a liquid sample containing analyte is mixed with reagent antigen coated fluorescent microspheres and larger microspheres coated with an antibody which bind specifically with the antigen. The particle suspension is measured using a laser flow cytometer for fluorescence and light scattering to provide data correlating to the analyte concentration in the sample. The particles are measured by fluorescence.
U.S. Pat. Nos. 4,588,680; 4,414,325; 4,503,143; 4,451,434; 4,703,017 and 4,458,014 describe methods of detecting analytes such as viruses, enzymes, microorganisms and other analytes where the analyte is bound to a particle and the analyte is detected by fluorescence or a color change.
U.S. Pat. No. 4,097,338 describes a method for determining a reduced coenzyme where the fluorescence of the reduced coenzyme is measured in an aqueous medium in the simultaneous presence of an organic liquid miscible with water and a dispersion of one or more slightly soluble or insoluble substances. The presence and combination of the organic liquid and particles enhances the fluorescence of the reduced coenzyme.
Canadian patent 1,103,137 discloses titration of an ion exchange colloidal polymer with an oppositely charged colloidal particle.
A surface-enhanced Raman spectroscopy technique for high pressure liquid chromatography (HPLC) and flow injection analysis (FIA) detection is described by Freeman et al, Applied Spectroscopy, 1988, 42:456. Analytes are adsorbed to colloidal silver and then the Raman spectrum is measured.
A reagent having an iron oxide nucleus and two layers of surfactants which can be used to magnetize particles and solid materials in non-aqueous environments is described in R & D Magazine, April 1992, p. 132.
In the methods described above where the particle is part of the detection process. Preconcentration of the particles does not decrease the detection limit because the background signal is also increased upon preconcentration. Processes in which the particular reagent dissolves in the analyte solution do not allow one to preconcentrate the sample. None of these methods allow one to effectively eliminate memory effects.
Ion-exchange resins are well known and extensively used to treat liquids and alter the chemical and physical properties of liquids. General uses of such resins include water softening (DE 1,294,933), pH control (U.S. Pat. No. 2,563,006), etc. carried out using a variety of apparatus, for example, those described in U.S. Pat. Nos. 4,900,434, 4,978,506, FR 1,577,527 and SU 671,828.
Another common use of ion-exchange resins is to chelate and preconcentrate trace elements in a sample for later analysis. A variety of chelating resins used to preconcentrate different analytes are described, for example, Horvath et al, Anal. Chem., 1986, 58:1352-1355; Horvath et al, Anal. Chim. Acta, 1985, 173:305-309; Koster et al, Anal. Chim. Acta, 1967, 38:179-184; Dumont et al, Appl. Spectr., 1989, 43:1132-1135; and Greenfield et al, Anal. Proc., 1989, 26:382-284. Soluble chelating compounds have also been used to extract and effect preliminary separation of analytes such as plutonium as described, for example, in Yu et al, Zhur. Anal. Khim., 1966, 21:1217-1222 (English translation).
Watson et al, S. Afr. J. Chem., 1984, 37:81-84 describes determination of trace noble metals by adsorption onto ion-exchange resin particles followed by direct injection into an ICP source. In this process, a specific ion-exchange resin was ground so as to pass through a 200 mesh screen (maximum grain size of 75 .mu.m). Platinum, palladium, ruthenium, rodium and gold analytes contained in leach residues were adsorbed onto the ion-exchange resin. A slurry is then formed from the resin with adsorbed metal analyte, the slurry is fed to a nebulizer and then to an ICP source. This method suffers from several disadvantages. Slurries are not stable suspensions and result in settling of large size particles within minutes. This type of slurry requires constant agitation to maintain the large particles in suspension. Further, use of large size particles to adsorb the analyte metals provides a non-uniform sample matrix for presentation to the ICP source. Use of a non-uniform sample matrix decreases the sensitivity of a detection method.
A need continues to exist, therefore, for improved analytical methods for detecting analytes. A need also exists for improved analytical methods for preconcentrating analytical samples, increasing sensitivity and analytical methods and eliminating memory effects.